HU211829A9

HU211829A9 - Haemophilus paragallinarum vaccine

Info

Publication number: HU211829A9
Application number: HU95P/P00562P
Authority: HU
Inventors: Antonius Arnoldus Chris Jacobs
Original assignee: Akzo Nobel Nv
Priority date: 1990-09-05
Filing date: 1995-06-29
Publication date: 1995-12-28
Also published as: JPH0592929A; CN1059471A; MX9100654A; DE69110997T2; HU210851B; AU640516B2; EP0475478B1; US5240705A; HUT61488A; IL99097A0; EP0475478A1; NZ239654A; CA2049129A1; HU912864D0; KR920006004A; ZA916237B; AU8347491A; DE69110997D1

Description

The present invention relates to a vaccine for the protection of poultry against infection by Haemophilus paragallinarum, an antigenic substance, anti-Haemophilus paragallinarum antigen, an antibody and antiserum thereof, and to a process for the preparation of a Haemophilus paragallinarum vaccine.

Haemophilus paragallinarum (H. paragallinarum) causes infection of the upper respiratory tract in chickens known as infectious rhinitis (IC). The most common symptoms are serous and mucous membrane of the sinuses and sinuses, facial edema and conjunctivitis. Infections of the lower respiratory tract, such as airbags and lungs, can also occur. Although morbidity is high, natural mortality is relatively low. ICs often cause an increase in cull numbers, stubborn growth and a decline in egg production, all of which result in significant economic losses. In addition, if the IC with other pathogens, e.g. pox, Mycoplasma gallisepticum, infectious bronchitis, Pasteurella, or infectious trachea, a more serious and prolonged disease that usually results in higher mortality. Birds recovered from IC often remain carriers of infection. and contribute to keeping the infection in the herd.

At present, vaccination against IC is carried out using whole bacterial vaccines. These vaccines are prepared from live strains, preferably attenuated strains or inactivated virulent strains. However, when using live attenuated vaccines, there is always a risk that the animals will be vaccinated with inadequately attenuated pathogenic bacteria, which will then cause the vaccinated animals to become ill and possibly spread the disease to the herd. In addition, the attenuated bacteria may return to virulence.

Inactivated vaccines also have a number of disadvantages, e.g. the appearance of side effects that may occur as a result of the administration of complete bacteria. that the materials of the cell wall, e.g. lipopolysaccharides (LPS) are present.

There are three distinct serotypes of H. paragallinarum, namely serotypes A, B and C. There is cross-protection between different strains of H. paragallinarum within each serotype. However, when a bird is vaccinated with a strain of H. paragallinarum belonging to a particular serotype, it is not protected against infection by strains of H. paragallinarum that belong to other serotypes, i.e. the immunity is type specific. Therefore, in order to provide adequate protection against IC, the birds should be immunized with a vaccine containing immunogenic material from several H. paragallinarium serotypes.

In addition to the said disadvantages of H. bacterialis vaccines based on whole bacteria, a further disadvantage of such vaccines is that a vaccine containing whole bacteria of serotype B or C only partially protects vaccinated birds against subsequent infection with serotype B or C virulent strains.

A fibrous hemagglutinin of H. paragallinarum serotype A was isolated and characterized by Iritani et al., Am. J. Vet. Gap. 41, 2114 (1980) and DEA2 906 996 (Shionogi & Co. LTD.)]. This fibrous hemagglutinin has a protective effect against H. paragallinarum serotype A infection.

However, the lithologic method for isolating the protective component of H. paragallinarum serotype A, wherein the protective activity is delivered from the cell using trypsin and isolating the carrier of protective activity, is not applicable to serotype C, since this isolate from H. paragallinarum strains a protective component would not be isolated during the session.

We have developed a new procedure for the isolation of the protective fraction of H. paragallinarum which is applicable to all H. paragallinarium serotypes.

This novel method yields a new purified membrane fraction from H. paragallinarum cells containing a 38 ± 3 kD protein (as measured by SDS-PAGE, i.e., sodium dodecylsulfate polyacrylamide gel electrophoresis as described below). which is highly protective against H. paragallinarum infection in poultry and is substantially free of cells and fibrous material.

In accordance with the present invention, a subunit vaccine is prepared, i.e., a vaccine containing only the protective and important components derived from the membrane fraction of H. paragallinarum cells, which contains the approximately 38 kD protein (38 kD protein enriched membrane). oil fractions). Chickens immunized with such vaccines are well protected against homologous infection with virulent strains of H. paragallinarum.

Even more complete protection can be achieved by administering to chickens a vaccine consisting of the outer membrane fraction of H. paragallinarum cells enriched in 38 kD outer membrane protein (OMP) (38 kD OMP formulation).

This purified 38 kD OMP formulation is essentially free of cells and fiber.

The vaccine according to the invention is further characterized by its protective activity

- heat sensitive,

sensitive to trypsin treatment and

- Sensitive to K-proteinase treatment.

The vaccine of the invention may also contain a fragment of said 38 kD OMP which still has immunizing properties, i.e., immunologically equivalent.

The membrane fraction enriched in the 38 kD protein is prepared by culturing H. paragallinarum bacteria in a suitable medium under conditions which promote expression of the 38 kD OMP, and then digesting the H. paragallinarum cells, e.g. by enzymatic or mechanical digestion (e.g., ultrasonic treatment, milling or French press), the cell membrane fraction is pelleted and the membrane fraction enriched in the 38 kD protein is separated, e.g. centrifugation.

The 38 kD OMP formulation can be isolated by

A9 to separate the above-mentioned membrane fraction into a further internal and external membrane fraction, e.g. density gradient with sedimentation or an internal membrane detergent, e.g. Based on differential solubilization of Triton with Χ-100 or sarcosine followed by centrifugation.

Alternatively, isolation of said fraction or preparation may be accomplished by the use of antiserum specific for 38 kD OMP (e.g., monoclonal antibody). The membrane extract of H. paragallinarum can be loaded onto a column pack containing antiserum specific for said 38 kD OMP, the adsorbed fraction of the extract is separated from the non-adsorbed material, and the adsorbed fraction is dissolved from the column pack.

If desired, the 38 kD OMP can be purified to homogeneity by methods known in the art, e.g. 38 kD OMP is solubilized by extraction from said 38 kD OMP composition with one or more detergents and further purified e.g. ion exchange or molecular sieve chromatography under conditions that do not affect the protective properties of the 38 kD OMP.

Purified fractions enriched in the 38 kD protein can be appropriately separated from known H. paragallinarum serotypes such as A, B, C and corresponding serotypes known in the art.

The 38 kD OMP to be used in the vaccine of the present invention can be prepared by chemical synthesis, purification from H. paragallinarum cells, or recombinant techniques.

In the latter case, nucleic acid sequences encoding said protein or fragments thereof are, e.g. may be identified by screening a genomic DNA library of H. paragallinarum for individual clones containing said sequences, e.g. specific reaction with polyclonal or monoclonal antibodies raised against 38 kD OMP. Nucleic acid sequences can be ligated to various expressing DNA sequences to give a so-called recombinant nucleic acid molecule with which to transform a suitable host. Such hybrid DNA molecules are e.g. plasmids or nucleic acids present in viruses. As a host cell, it is of prokaryotic origin, e.g. of bacterial or eukaryotic origin, e.g. mammalian cells. The transformed host cells can be used to produce 38 kD OMP, and then the material containing said protein or 38 kD OMP can be isolated and used in the present invention to produce a vaccine.

In another embodiment, a live vector vaccine may be prepared comprising a non-pathogenic microorganism, such as a virus or bacterium, wherein the nucleic acid sequence encoding the 38 kD OMP or fragment thereof is cloned into the microorganism.

The vaccines of the present invention comprise a membrane fraction enriched in 38 kD protein or a 38 kD OMP formulation of at least one serotype of H. paragallinarum, i.e., vaccines comprising such fraction or formulation, or vaccines comprising the 38 kD protein. s containing an immunological equivalent of OMP are included within the scope of the invention.

The vaccine of the present invention may also be in the form of a recombinant DNA vaccine, as outlined above.

Preferably, the vaccine of the present invention comprises more than one 38 kD protein enriched membrane fraction or 38 kD OMP composition from H. paragallinarum strains of different serotypes. The most complete protection against H. paragallinarum is achieved by administering to the birds a vaccine containing a membrane fraction or composition derived from H. paragallinarum serotype A, B and C strains. Such a trivalent vaccine is a preferred embodiment of the present invention. Vaccination with the trivalent vaccine of the present invention fully protects birds against H. paragallinarum infections, in contrast to the full bacterial vaccines used hitherto.

In addition to the H. paragallinarum protective carrier, the vaccine of the invention may contain an aqueous medium or a suspension medium containing water, often in admixture with other ingredients, for example to increase activity and / or shelf life. These ingredients may include salts, pH buffers, stabilizers (e.g., skim milk or casein hydrolyzate), emulsifiers, adjuvants to enhance the immune response (e.g., mineral oils, muramyl dipeptide, aluminum hydroxide, saponin, polyanions, and amphipathic compounds). . thimerosal.

The vaccine of the invention may also contain other immunogenic material derived from bacterial or viral infections in poultry, e.g. Mycoplasma, an immunogenic substance of Newcastle disease virus or infectious bronchitis virus.

The vaccine is preferably administered parenterally, e.g. administered subcutaneously or intramuscularly. The vaccine can thus be administered both for the active immunization of vaccinated birds and for the passive immunization of the offspring of laying birds. Of course, in immunized laying birds, the antibodies they produce enter the yolk of their eggs, and thus the hatched chickens.

Both the composition of the vaccine and the vaccination system may be varied depending upon the type of animal to be protected, the age and weight of the animal, the desired duration of protection and the route of administration, and whether active immunization or passive immunization by maternal antibodies is desired. The optimum effective amount of the active component in the vaccine for parenteral, for example, intramuscular or subcutaneous vaccination of poultry, is about 0.1 to 100 pg, most preferably 0.3 to 1.0 pg of protein per dose.

Said active immunization against H. paragallinarum

EN 211 829 A9 is used primarily as a protective treatment for healthy birds. It will be appreciated that birds already infected with H. paragallinarum may be treated with antibodies against the membrane fraction or composition of the invention.

Antiserum against the membrane fraction of the present invention is prepared by immunizing birds with an effective amount of said fraction, preferably in the presence of an adjuvant. If desired, the animals are vaccinated with a second vaccination to induce an appropriate immune response. The animals are then bled and antiserum is prepared from the blood.

The antiserum or antibodies may be mixed with pharmaceutically acceptable carriers.

A polyclonal antiserum specific for a membrane fraction from a strain of H. paragallinarum of a particular serotype may be prepared by incubating the antiserum against said membrane fraction with a mixture of membrane fractions of another strain of H. paragallinarum serotype. Antibodies that are not specific to the said H. paragallinarum membrane fraction are adsorbed to the added H. paragallinarum fractions and can be separated from the incubation mixture to give an antiserum composition specific for a membrane fraction of a particular H. paragallinarum serotype.

Monoclonal antibodies to the membrane fraction or composition of the invention may also be used to passively immunize birds infected with H. paragallinarum. Said monoclonal antibodies can be prepared in a known manner, for example, by immunizing mice with the membrane fraction of the invention, immortalizing mouse spleen cells, and selecting hybridomas that produce suitable antibodies.

Antisera and monoclonal antibodies can also be used to immunologically diagnose birds infected with H. paragallinarum bacteria.

Example 1

Purification of H. paragallinarum membrane fractions and immunization of chickens

Bacterial strains were plated on chocolate agar and incubated for 24 hours at 37 ° C in an atmosphere in which a candle was left to burn and cultured in TPB supplemented with 0.01% NADH.

230 ml of concentrated cell suspension of HI 18 (serotype C) [containing 4x10 ¹⁰ cells / ml in PBS containing 40 mM PBS containing 0.01% thimerosal was sonicated for 6 minutes at 4 ° C with 100 W, Branson B12 Sonifier device (alternately 30 seconds ultrasonic treatment and 30 seconds cooling). Subsequently, uncomminuted cells were removed by centrifugation (10 min, 5000 g) and 20 mL of supernatant was emulsified in petroleum water-in-oil (W / 0) adjuvant. This vaccine is called SUP-USO (C). The remaining 210 ml of supernatant was centrifuged in an ultracentrifuge at 167,000 g for 1 hour to purify the total membrane fraction from the soluble fraction.

The membrane pellet (brownish) was resuspended in 60 ml of 50 mM Tris.HCl buffer, pH 8.0. 10 ml of this suspension was diluted three times with the same buffer and emulsified with W / 0 adjuvant. This vaccine is referred to as the membrane fraction (C) rich in 38 kD protein. The remaining 50 ml of the suspension was mixed with a buffer containing 50 mM Tris.HCl (pH 8.0) and 2% sodium N-lauroyl sarcosine in a 1: 1 ratio and incubated for 2 hours at room temperature to give the inner membranes. to solubilize. Subsequently, the mixture was centrifuged again at 167,000 g for 1 hour to separate the solubilized inner membranes from the outer membranes.

The pellet was resuspended in 50 mL of 50 mM Tris.HCl, pH 8.0. A portion of this suspension is then diluted three times with the same buffer and emulsified with W / O adjuvant. This vaccine (C) is called a 38 kD OMP formulation.

The protective capacity of the various purified W / O adjuvanted fractions is shown in Table 1,

Commercial Isabrown chickens were vaccinated once with 10 ml of various vaccines at the age of 10 weeks and challenged four weeks later with H. paragallinarum strain H18 (serotype C). Six chickens were used for each vaccine.

Table I

Protection of chickens by immunization with purified fractions against H. paragallinaru infection

vaccine clinical signs Reisolation score ³ % protection the number of positive chickens is ⁶ % protection control 17 0 6 0 SUP-USO (C) 12 29 4 33 Membrane fraction rich in 38 kD protein (C) 1 94 1 83 38kD OMP Preparation (C) 0 100 0 100

³ The score represents the total number of chickens with nasal discharge from 3 observations: 24 hours, 48 hours and 5 days after infection ⁶ Re-isolation from infraorbital cavity 5 days after infection (satellite growth)

As shown in Table 1, the purified membrane fraction of H. paragallinarium, and in particular the purified 38 kD OMP (C) formulation,

A9 needles according to Laemmli (Naturre 227: 680-685 (1970)).

SDS-PAGE of the purified formulations together with CBB staining revealed a band of approximately 38 kD as the dominant protein in OMP formulations (Fig. 1). Figure 1 shows the SUP-U50 and 38 kD of the different formulations, i.e. strain 083 (A) (AB lines), Spross (B) strain (DE lines) and H-18 (C) strain (GH lines). shows SDS gel electrophoresis profiles of OMP preparations. In lines C, F and I, the molecular weight

round can be found: Cytochrome C, horse origin 12.3 kD Myoglobin, of horse origin 17.2 kD Carbonic anhydrase, bovine 15 DETU 30.0 kD Ovalbumin, made from hen's egg 43.0 kD Album from bovine serum 66.3 kD Ovotransferrin from hen eggs 78.0 kD The protective activity of the 38 kD OMP formulations is to-

To determine its further characteristics, the compositions were purified as described in Example 1 from strains 083 (A), Spross (B) and H-18 (C) and subjected to heat treatment and enzymatic digestion (trypsin and K proteinase).

Trypsin cleaves proteins after positively charged amino acid residues (i.e., lysine, arginine). K-proteinase is a strong enzyme and cuts most proteins into small peptides. Heat treatment to 100 ° C leaves the amino acid chain at 5 inches intact, but destroys the tertiary and quaternary structure of most proteins (mostly irreversibly). For enzymatic digestion, 2 mg of protein was incubated with 200 pg of enzyme in 1 ml of 40 mM PBS for 1.5 hours at 37 ° C. Control preparations were treated in the same manner except that the enzyme was omitted. For the production of heat denatured proteins, 2 mg of protein was heated in 1 ml 40 mM PBS at 100 ° C for 10 minutes.

Vaccine compositions containing 50 µg of protein in a 0.5 ml dose of 0 / W emulsion were prepared and tested for homologous infection. Commercially available 10-week-old Isabrown chickens were vaccinated once and infected 4 weeks later. 6-6 chickens were used in each infection group (Table 4).

From the experiments it can be concluded that protective activity is sensitive to heat and enzymatic treatment since such treatments resulted in a strong decrease in protective capacity in all three serotypes.

Table 4

Effect of enzymatic and heat treatment on the protective effect of 38 kD OMP preparations.

Vaccine Type A Infection Type B Infection Type C Clinical Signs ³ Re-Isolation ⁶ Clinical Signs ³ Re-Isolation ⁶ Clinical Signs ³ Re-Isolation ⁶ OMP Treatment Score%

Claims

A vaccine for protection of poultry against Haemophilus paragallinarum infection, characterized in that the vaccine is essentially cell-free and is derived from the membrane fraction of Haemophilus paragallinarum, which is approximately 38 (+ / 3) kD, measured by SDS-PAGE. -fibrous protein or an immunological equivalent thereof.

Vaccine according to claim 1, characterized in that it is derived from an outer membrane composition comprising the 38 kD protein as the major protein component.

The vaccine according to claim 1 or 2, characterized in that it is derived from membrane fractions of at least two different serotypes of Haemophilus paragallinarum.

Vaccine according to claim 3, characterized in that it is derived from membrane fractions of Haemophilus paragallinarum serotype A, B and C cells.

The A9 appeared to be potent, probably due to the optimal immune response elicited by said fraction.

To prepare a monovalent 38 kD OMP (A), strain 083 (A) was plated on chocolate agar and incubated for 24 hours at 37 ° C in a candle-fired atmosphere and cultured in TPB supplemented with 0.01% NADH. . After incubation at 37 ° C for 24 hours, cells were centrifuged and purified 38 kD outer membrane protein (A) was isolated as described for 38 kD OMP (C).

Isolated OMP (A) was emulsified with W / 0 adjuvant to give vaccines containing 10 µg / 0.5 ml and 1 µg / 0.5 ml of active ingredient, respectively. To test these vaccines, commercially available 10-week-old Isabrown chickens were vaccinated intramuscularly (breast) with a single dose of 0.5 ml. On the day of vaccination and 4 weeks after vaccination, blood samples were taken and the sera were assayed for antibodies to inhibit hemagglutination (HI) (antibodies which are indicative of protection) by known methods.

Table 2

Hl-A antibody titers in chickens after vaccination with 38 kD OMP (A)

Vaccine The dose HI-A titer ³ 10 gg 7 5 8 7 10 1 gg 5 5 8 7 8 control 0 0 0 0 0

² log titers, measured 4 weeks after vaccination with 5 chickens per puff

From the results (Table 2), it can be concluded that 100% of the chickens vaccinated at 10 and 1 gg were protected (HIA> 2 ”).

Monovalent 38 kD OMP (B) was prepared in exactly the same manner as described above except that Spross (B) was cultured and used as starting material to isolate the 38 kD OMP.

Example 2

Protection of chickens immunized with a trivalent vaccine

In one experiment, a trivalent subunit vaccine (in W / 0 adjuvant) was prepared and tested for protection against serotype A, B, or C strains.

In addition, a monovalent H-18 (C) vaccine was tested against heterologous infection with strain 083 (A) and Spross (B). The 38 kD OMP preparations of strain 083 (A), Spross (B), and strain H-18 (C) used to prepare the trivalent subunit vaccine were purified in the same manner as described in Example 1. The trivalent vaccine contained 100 µg of each protein per 0.5 ml dose. The monovalent H-18 (C) vaccine is identical to a

38 kD OMP formulation with the vaccine used in Example 1.

Commercially available 10-week-old Isabrown chickens were vaccinated once with 0.5 ml of the vaccine and infected 4 weeks later. 7-7 chickens were used for each infection group.

Table 3

Protective effect of a trivalent subunit vaccine and a monovalent (type C) subunit vaccine

infectious tribe (serotype pus) vaccine clinical signs Reisolation score ^a % protection number of positive chickens ¹ ' % protection 083 (A) control 18 0 7 0 083 (A) trivalent 0 100 0 100 1645 (THE) control 18 0 7 0 1645 (THE) trivalent 0 100 0 100 Spross (B) control 19 0 7 0 Spross (B) trivalent 0 100 0 100 H-I8 (C) control 20 0 7 0 H-18 (C) trivalent 0 100 1 86 Modes- to (C) control 20 0 7 0 Modes- to (C) trivalent 21 100 1 86 083 (A) monova- lens 21 0 7 0 Spross (B) monova- lens 19 0 7 0

³ The score represents the total number of nasal chicks from 3 observations; 24 hours, 48 hours, and 5 days after infection ⁶ Re-isolation of the intra-abrasive cavity bit 5 days after infection (satellite growth)

Table 3 shows that only homologous protection is induced after vaccination with the monovalent H-18 (C) vaccine. The trivalent vaccine provides complete protection against infection with all strains tested.

Example 3

Characterization of membrane fractions rich in 38 kD protein

The membrane fractions rich in the 38 kD protein and the 38 kD OMP preparations derived from H. paragallinarum serotypes A, B, and C (strain 083, Spross strain, and H-18 strain) were purified as described in Example 1.

Purified formulations are run on SDS-PAGE5

HU 211 829 A9

5. Vaccine according to any one of claims 1 to 3, characterized in that it contains at least one further microorganism infecting poultry or an immunogenic material thereof.

6. Vaccine according to any one of claims 1 to 5, characterized in that it also contains an adjuvant.

7. Haemophilus paragallinarum antigen, characterized in that it has a membrane fraction of Haemophilus paragallinarum comprising about 38 (+/- 3) kD of non-fiber protein or 10 immuno equivalents thereof, as measured by SDS-PAGE, and substantially free of cells and fibrous material.

The antigenic material of claim 7, wherein said outer membrane composition comprises a 38 kD protein as a major protein component.

An antibody or antiserum characterized in that it is immunoreactive with the Haemophilus paragallinarum antigen according to claim 7 or 8.

10. A pharmaceutical composition comprising: a

The antibody or antiserum of claim 9.

11. A process for the preparation of a vaccine according to claim 1, characterized by culturing Haemophilus paragallinarum bacteria in a culture medium, extracting its membrane material containing 38 (+/- 3) kD non-fibrous protein and processing it into an immunizing activity formulation. .